【摘要】 目的探讨用体外转录方法制备EBV-LMP1C端RNA并用该RNA对其靶脱氧核酶进行初步筛选。方法用巢式PCR方法从绒猴淋巴细胞系B95-8细胞中扩增EBV-LMP1exonc,重组入pGEM-11zf载体,并用T7RNA聚合酶对其进行体外转录制备EBV-LMP1C端RNA。根据该RNA的一、二级结构设计合成3种靶向10～23脱氧核酶,据体外剪切效率筛选高效特异的靶向脱氧核酶。结果成功地构建含EBV-LMP1exonc基因的重组质粒,用体外转录方法用1μg质粒转录出40.25μg高纯度的EBV-LMP1C端RNA,有1种脱氧核酶DZ1对该RNA体外剪切效率达89%。结论用T7RNA聚合酶体外转录方法成功制备了高纯度的EBV-LMP1C端RNA,经体外剪切筛选出高效特异的1种脱氧核酶,为进一步研究奠定了基础。更多还原

【Abstract】 Objective To synthesize highly pure EBV-LMP1 carboxy region RNA by in vitro transcription and select its targeted active DNAzyme.Methods EBV-LMP1 exon c gene was amplified by nest PCR from the B95-8 cell genomic DNA and cloned into plasmid pGEM-11zf.The cloned DNA sequence was transcribed in vitro by T7-RNA polymerase.Three targeted 10-23 DNAzymes were designed and synthesized.The highly active and specific DNAzymes were evaluated by in vitro cleavage reaction.Results EBV-LMP1 exon c gene recombinant plasmid was successfully constructed and 40.25 μg EBV-LMP1 carboxy region RNA was produced via transcription in vitro from 1.0 μg lined recombinant plasmid.The cleavage ratio of active DNAzyme DZ1 was 89%.Conclusion Highly pure EBV-LMP1 carboxy region RNA was synthesized successfully by in vitro transcription.One active targeted DNAzyme was selected by in vitro cleavage reaction,paving the way for further research.更多还原